Karyotaxonomy of Myosotis Alpestris Group

Preslia 78: 345–352, 2006 345

Karyotaxonomy of Myosotis alpestris group

Chromozomové počty a taxonomické poznámky k zástupcům skupiny Myosotis alpestris

Jitka Š t ě p á n k o v á

Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Průhonice, e-mail: [email protected]

Štěpánková J. (2006): Karyotaxonomy of Myosotis alpestris group. – Preslia 78: 345–352.

Chromosome numbers of taxa belonging to the Myosotis alpestris group are provided and/or confirmed. A chromosome count is reported for the first time for M. olympica. A new ploidy level (2n = 24) was revealed within M. stenophylla for which previously only tetraploid cytotypes are reported. Myosotis stenophylla is identified for the first time from Greece. Previous chromosome counts for M. ambigens, M. alpestris, M. atlantica, M. corsicana, M. lithospermifolia, and M. suaveolens are confirmed based on plants originating from karyologically poorly investigated parts of the distribution areas of this polyploid complex.

K e y w o r d s : distribution area, karyogeography, Morocco, Myosotis alpestris group, ploidy level, South Europe, taxonomy, Turkey

Introduction Representatives of the Myosotis alpestris group are arcto-alpine species with conspicuously scattered distribution areas. The population system of island differentiation is characteristic of this complex; suitable habitats are relatively small but scattered over a large region. Thus, a typical variation pattern develops as a result of limited gene flow, selection pressure of the environment and genetic drift. Along with morphological variation, extensive variability in ploidy level is found in this group. Several factors acting interactively must be invoked to ex- plain the considerably complicated pattern of variability within the M. alpestris group. The most important ones are: polyploidy, ecological adaptation and geographical isolation. In the Myosotis alpestris group, significant chromosome variation among species occurs. Karyological studies of this group have been published by several authors. Of particular in- terest are reports by Grau (1964), Luque (1992) and Štěpánková (1993a, 1993b, 1996). So far three groups of alpine forget-me-nots can be recognized based on their ploidy level: diploids (2n = 24), tetraploids (2n = 48) and hexaploids [2n = 72 (70)]. It is quite clear that the process of polyploidization has greatly contributed to the evolution and speciation within this group. Along with conspicuous karyological variation and geographic discontinuities, the species of this group are well known for their interesting ecological disjunction. They grow on serpentine as well as non-serpentine sites both at alpine and lower altitudes. Hence the M. alpestris group might serve as a model for studies of the origin and consequences of geographic and ecological endemism, polyploid evolution and biogeographic history of the European mountain flora. However, the modes of diversification in the group are still un- clear and no phylogenetic hypotheses are proposed. The results of karyological examination presented here are the first part of the project focussed on the biogeographic and evolution- ary history of the complex, combining molecular phylogeny (data from restriction site 346 Preslia 78: 345–352, 2006 analysis of cpDNA and sequence data of ITS region of rDNA) with phylogenetic informa- tion from non-molecular data sets (morphology, karyology).

Material and methods Chromosome numbers were counted in samples from 21 populations collected in the field (Table 1) and cultivated in the experimental garden of the Institute of Botany at Průhonice. Counts were usually made on three different root tips from each of two to five individuals cho- sen from each population. Chromosome numbers were determined at the mitotic metaphase in the somatic cells of root tips. The material was pretreated for two hours with a saturated solu- tion of bromnaphtalen and fixed in a freshly prepared 3 : 1 mixture of ethanol and acetic acid (Carnoy). The squash method and staining by lacto-propionic orceine were used. Voucher specimens are deposited in the herbarium of the Institute of Botany at Průhonice (PRA).

Results and discussion Myosotis alpestris F. W. Schmidt A species distributed in almost all high mountain ranges in Europe and having broad ecological preferences with respect to the geological substrate. Its diagnostic set of morphological characters is: basal leaves with oblanceolate to obovate blade gradually narrowing into a petiol, hirsute with more or less patent hairs or glabrescent on lower surface, calyx narrowed at the base in fruit, with straight, short, appressed hairs not forming conspicuous white rim at the margin of calyx teeth, with few longer, patent, slightly curved hairs and sometimes with few hooked stiff hairs on the calyx tube, not extending up to the flower/ fruit pedicel (Fig. 1a, b), pedicel usually as long as calyx in fruit. Myosotis alpestris is well known for its ability to form cytotypes of different ploidy levels. However, this variation in ploidy level is not associated with particular morphological characters. According to previously reported chromosome data (e.g. Grau 1964, Štěpán- ková 1993b), diploid and tetraploid cytotypes occur sympatrically in the Pyrenees, Alps and Carpathians. Hexaploids are known only from the Alps in Germany and Austria (Grau 1964, Štěpánková 1993b). Tetraploid chromosome counts based on plants from the Velebit Mts (Croatia), Prokletije Mts (Montenegro and Albania), Mt Kajmakčalan (Greece), and Pindos Mts (Greece) are reported here for the first time. During the revision of the M. alpestris group in Europe, a noteworthy discrepancy in taxonomic evaluation of sympatric diploid/polyploid cytotypes was noticed. While diploids, tetraploids, and hexaploids occupying areas of the Alps and the Carpathians are not usually classified at any taxonomic level (e.g. Grau 1964, Grau & Merxmüller 1972), diploids and tetraploids from the Pyrenees are usually treated as separate taxonomic units (Grau 1964, Grau & Merxmüller 1972, Blaise et al. 1992). While the tetraploids are attributed to M. alpestris, diploids are treated as M. pyrenaica (= M. alpina Lapeyer., nom. illeg.; Grau 1964, Grau & Merxmüller 1972), M. corsicana subsp. pyrenaeorum (Blaise et al. 1992), or M. alpestris (Luque 1992). Plants collected in the Pyrenees and included in this karyological analysis bear all of the diagnostic characters of M. alpestris, hence the diploid chromosome number of 2n = 24 reported here is attributed to M. alpestris. Štěpánková: Karyotaxonomy of Myosotis alpestris group 347

Table 1. – List of localities and chromosome numbers of the populations ofMyosotis studied.

Taxon Voucher 2n Collection locality Coordinates Altitude Collectors number (m a.s.l)

M. atlantica 2006/1 24 Morocco: Haute Atlas Mts, 31°13' N 2550 Štěpánková J., Oukaïmeden 07°51' W Kaplan Z., Kirschner J. 2002 M. alpestris 2005/1 24 Spain: Pyréneés-Orientales 42°41' N 2483 Štěpánková J., Mts, Puerta da la Bonaiqua, 00°59' E 2005 Mt Tuc de la Llanca M. alpestris 2005/2 48 Spain: Pyréneés-Orientales 42°36' N 2380 Štěpánková J. Mts, Parc National de Aigües 00°58' E 2005 Tortes, Port de Ratera M. alpestris 2005/3 48 France: Pyréneés-Orientales 42°34' N 2059 Štěpánková J. Mts, Mt Pic Carlit 01°54' E 2005 M. alpestris 2005/4 48 Montenegro: Prokletije Mts, 42°31' N 1750 Štěpánková J. Gusinje, Mt Volušnica 19°46' E 2005 M. alpestris 2005/5 48 Greece: Northern Pindos Mts, 39°40' N 1640 Štěpánková J. Metsovo, Kataras pass 21°21' E 2003 M. alpestris 2005/6 48 Greece: Nidže voras Mts, Mt 40°54' N 1886 Štěpánková J. Kajmakčalan. 21°49' E 2003 M. alpestris 2005/7 48 Croatia: Velebit Mts, Mt 44°46' N 1620 Štěpánková J. Crikvena 14°59' E 2005 M. ambigens 2005/8 24 Italy: Monte Baldo Mts, the 45°38' N 1500 Štěpánková J., crest Creta di Naole 10°47' E Kaplan Z. 2004 M. corsicana 2005/9 24 Corsica: Vizzavona, Mt Monte 42°08' N 2203 Šafránek J. D’Oro 29°06' E 2004 M. lithospermifolia2005/10 24 Turkey: Esler Dagi Mts, Mt 37°40' N 1950 Štěpánková J. Honaz Dag 29°18' E 2004 M. lithospermifolia2005/11 24 Turkey: Dedegöl Daglari Mts, 37°41' N 2036 Štěpánková J. Mt Dededöl Dagi 31°18' E 2004 M. lithospermifolia2005/12 24 Turkey: Ak Daglar Mts: Mt 36°31' N 2108 Štěpánková J. Kofu Tepe 29°55' E 2004 M. olympica 2005/13 24 Turkey: Uludag Mts, Mt Uludag40°05' N 2232 Štěpánková J. Tepe 29°11' E 2004 M. stenophylla 2005/14 24 Greece: Northern Pindos Mts, 40°06' N 2087 Štěpánková J. Mt Smolikas 20°54' E 2003 M. stenophylla 2005/15 24 Greece: Northern Pindos Mts, 40°05' N 2600 Štěpánková J. Mt Smolikas 20°55' E 2003 M. suaveolens 2005/16 24 Greece: Central Olympus Mts, 40°05' N 2815 Štěpánková J. Mt Skolio 22°22' E 2003 M. suaveolens 2005/17 24 Montenegro: Prokletije Mts, 42°30' N 1790 Štěpánková J. Gusinje, Mt Karanfili 19°47' E 2005 M. suaveolens 2005/18 24 Albania: Prokletije Mts, Mt 42°27' N 2308 Štěpánková J. Maja e Jezerce 19°49' E 2005 M. suaveolens 2005/19 24 Montenegro: Komovi Mts: 42°43' N 2100 Štěpánek J., Jr. Berane, Mt Kom Vasojevički 19°41' E 2005 M. suaveolens 2005/20 24 Montenegro: Moračke Planine 42°50' N 1840 Štěpánková J. Mts: Berane, Mt Moračka Kapa 19°15' E 2005 348 Preslia 78: 345–352, 2006

Myosotis ambigens (Bég.) Grau A species considered to be restricted to the Apennine Mts (Italy) (Grau 1964, Grau & Merxmüller 1982). They are small mat-forming plants with small short petiolate basal leaves and narrowly oblanceolate blade hirsute with long patent hairs on both surfaces or sometimes glabrescent on the lower surface, gradually narrowing into a petiole, with conspicuously smaller linear to lanceolate hirsute cauline leaves (ca 5 × 15 mm in the middle part of stem), obtuse at the apex, with fruiting calyx more or less narrowed at the base, densely covered with short hairs and with numerous long stiff appressed white hairs, forming conspicuous white rim at the margin of calyx teeth, calyx tube with no or very few hooked hairs, pedicel without hooked hairs (Fig. 1c). During an excursion to the small mountain massif of Monte Baldo Mts (Italy, Veneto, near Lake Garda) small populations of plants with these characters were found. These plants were identified as M. ambigens on the basis of a detailed comparison with plants of M. ambigens from the Central Apennines (La Maiella Mts and Gran Sasso Mts). The chromosome number found agrees with that indicated by Grau (1964) and Blaise & Cartier (1982) for plants of M. ambigens coming from the Central Apennines.

Myosotis atlantica Vestergr. This taxon is endemic to the high mountain systems of Morocco. It is distinct from the other species of this group as all its leaves are pilose with long soft nearly straight hairs on both surfaces, calyx rounded at the base in fruit, with white straight hairs forming conspicuous white rim at the margin of calyx teeth and with longer, patent, slightly curved hairs and hooked ones on the calyx tube extending halfway up the pedicel. Patent hooked hairs on the flower/fruit pedicels is a noteworthy morphological character, which occurs very rarely within the M. alpestris group (Fig. 2a). Myosotis atlantica, M. corsicana and M. olympica are the only representatives with this character. The chromosome count presented here confirms the diploid level (2n = 24) previously reported by Blaise (1970) and Galland (1988).

Myosotis corsicana (Fiori) Grau A rare and local endemic of Corsica (France), M. corsicana, is an attractive perennial species occupying the highest parts of the central and northern mountains of the island. It dif- fers from other representatives of the group M. alpestris by having strigose leaves with retrose-appressed, short stiff hairs scattered on upper surface, glabrate or glabrescent lower surface, basal leaves long petiolate with blade broadly obovate or elliptical, more or less rounded at the base and suddenly constricted into a long petiole, cauline leaves coriaceous, broadly ovate, subacute at the apex, calyx rounded at the base in fruit, with straight, short, appressed hairs and long hairs on the calyx teeth forming conspicuous white rim, calyx tube with long patent or slightly decumbent hooked stiff hairs, extending up to the flower/fruit pedicel (Fig. 2b), pedicel not longer than calyx in fruit. The chromosome count of 2n = 24 was observed in plants from Monte d’Oro. This number agrees with that previously reported by Countandriopoulos (1964). Štěpánková: Karyotaxonomy of Myosotis alpestris group 349

c d

Fig. 1. – Hairs on the base of calyx: a – M. alpestris (diploid), Alps, Italy; b – M. alpestris (diploid), Pyrenees, Spain; c – M. ambiguens,MtGranSasso,Italy;d–M. stenophylla, Czech Republic, Mohelno. Scale bar = 500 μm.

Myosotis lithospermifolia (Willd.) Hornem. The distribution area of this species includes Turkey, Iraq, Iran, Crimea and the Caucasus (Grau 1964, 1978). Its diagnostic characters are densely pilose leaves with long soft hairs on both surfaces, basal leaves indistinctly petiolate with oblanceolate to narrowly obovate blade, obtuse at apex, cauline leaves narrowly lanceolate, subacute to obtuse at apex, calyx narrowed at the base in fruit, equally densely hairy with straight, short, appressed hairs and long hairs on the calyx teeth not forming conspicuous white rim, calyx tube densely covered by long patent to deflexed hooked stiff hairs, extending halfway up the flower/fruit pedicel (Fig. 2c), fruit pedicels conspicuously longer than fruiting calyx. The chromosome number of 2n = 24, found in the three populations of M. lithospermifolia, is the first count for Turkey. This chromosome number agrees with the one previously established by Grau (1964) for plants from Iraq. Based on these reports, M. lithospermifolia seems to be a diploid with 2n = 24. 350 Preslia 78: 345–352, 2006

a b

c d

Fig. 2. – Hairs on the base of calyx: a – Myosotis atlantica, Haute Atlas Mts, Morocco; b – M. corsicana, Corsica; c–M. lithospermifolia, Mt Dedegöl Dagi, Turkey; d – M. olympica, Mt Uludag Tepe, Turkey. Scale bar = 500 μm.

Myosotis olympica Boiss. This species is described from the Turkey, Mt Olympi Bithyni (recently Mt Uludag Tepe) near Bursa and recorded from other high mountains of Turkey, predominantly the Pontic mountain systems (Grau 1978). The diagnostic characters of M. olympica include strigose leaves with short stiff hairs on the upper surface, glabrate or glabrescent lower surface, blade of basal leaves elliptical or obovate, subacute at apex, at base very gradually narrowing into a petiole, cauline leaves narrowly ovate to lanceolate, subacute at apex, fruiting calyx narrowed at base, with long, adpressed, straight hairs on calyx teeth forming distinct white rim at the margins and conspicuous white wad between teeth, calyx tube with long hooked hairs extending halway up the flower/fruit pedicel (Fig. 2d), pedicel usually shorter than calyx in fruit. Karyological examinations of the locus classicus population revealed a diploid chromosome number 2n = 24. As far as the author knows, this is the first report for this species. Štěpánková: Karyotaxonomy of Myosotis alpestris group 351

Myosotis stenophylla Knaf While most of the taxa belonging to the Myosotis alpestris group are considered to occur in alpine sites of high mountains systems, M. stenophylla is the exception; so far it is only recorded from lowland Central Europe. The distribution of this forget-me-not shows an interesting ecological disjunction. It grows on serpentine as well as non-serpentine sites at lower altitudes. During an excursion to the North Pindos Mts (Greece), plants of M. stenophylla were found by the author on serpentine slopes of Mt Smolikas. There are several populations in the upper parts of the mountain (ranging from 2080 to 2600 m a. s. l.). Plants from Mt Smolikas have all the important features of M. stenophylla growing on serpentine sites: small plants with stem highly branched, basal leaves with long slender peti- oles and elliptical to oblanceolate blades, sparsely hirsute or glabrescent upper surface, glabrate to glabrescent lower surface, cauline leaves narrowly lanceolate, sparsely hairy or glabrescent on both surfaces, corolla conspicuously glowingly blue, calyx narrowed at base in fruit, with straight, short, appressed hairs not forming conspicuous white rim at the margin of calyx teeth, with longer, more or less patent, hairs both on the teeth and calyx tube, calyx tube sometimes with a few short hooked hairs not extending up to the flower/fruit pedicel (Fig. 1d), pedicel as long as calyx in fruit or a little longer. The chromosome count of 2n = 24 recorded for two populations from Mt Smolikas is a new diploid chromosome number for this species. Up to now, Myosotis stenophylla has been considered to be a tetraploid species (e.g. Štěpánková 1993b, 1996). However, a morphological comparison of tetraploid populations originating from lowland serpentine localities in Central Europe with diploid population from Mt Smolikas did not reveal any noteworthy difference. Hence, diploid populations from Mt Smolikas played very important role in the phylogeny of M. stenophylla.

Myosotis. suaveolens Waldst. et Kit. ex Willd. Together with M. alpestris, M. suaveolens is the most common representative of the M. alpestris group in mountain areas of the Balkan Peninsula. Diagnostic set of characters is: leaves on both surfaces densely hirsute with more or less patent soft hairs, basal leaves with oblanceolate to obovate blade gradually narrowing into a short, sometimes indistinct petiole, cauline ones ovate, calyx narrowed at the base in fruit, densely covered by straight, short, appressed hairs not forming conspicuous white rim at the margin of calyx teeth, with longer, patent, slightly curved hairs and with many hooked stiff hairs on the calyx tube, not extending up to the pedicel, pedicel shorter than calyx in fruit. Two ploidy levels, diploid and tetraploid, are recorded for this taxon. The chromosome number 2n = 24 is reported for M. suaveolens (Grau 1964, Greece, Akarnanika Mts, Boumistos; Štěpánková 1993a, Bulgarija, Rila Mts). The same count is recorded by Krogulevich (1978) for material from the East Sayan Mts (Vostočnyj Sajan). However, the taxon Myosotis suaveolens has been considered to be restricted to the Balkan Peninsula and its occurrence in the Sayan mountain range would require a taxonomic revision. Based on material from Northern Greece (Kozani), the only tetraploid chromosome number 2n = 48 is reported by Grau (1964). All the counts made by the present author confirm the diploid level (2n = 24) for this species. 352 Preslia 78: 345–352, 2006

Acknowledgements I am especially grateful to Zita Červenková, Jan Štěpánek Jr. and Luděk Štěpánek for their considerable help during my field work. My sincere thanks are due to Jan Kirschner and Zdeněk Kaplan for including me in the staff of the expedition to Morocco. I thank Tony Dixon for improving my English. This project was supported by grant no. IAA60053 of the Grant Agency of the Academy of Sciences of the Czech Republic and by grant of Academy of Sciences of the Czech Republic no. AV0Z60050516.

Souhrn V práci jsou uvedeny výsledky karyologické analýzy druhů Myosotis alpestris, M. ambigens, M. atlantica, M. corsicana, M. lithospermifolia, M. olympica, M. stenophylla a M. suaveolens. Pro druh M. olympica byl poprvé stanoven chromozomový počet. Bylo zjištěno, že tento druh je diploidní s chromozomovým počtem 2n = 24. Nově byl pro území Řecka zjištěn druh M. stenophylla. Zajímavá populace tohoto druhu známého jen z nižších poloh střední Evropy byla nalezena na hadcových svazích nejvyšší hory Severního Píndosu (severní Řecko) ve výšce od 2080 m až po vrchol (ca 2600 m). Populace je významná nejenom svojí izolovanou polohou a vysokou nadmořskou výškou lokality, ale zejména zjištěným chromozomovým počtem. Poprvé byl u tohoto druhu stanoven diploidní počet 2n = 24. Z tohoto hlediska je řecká diploidní populace rostoucí na hadcovém substrátu vý- znamným článkem v evoluci druhu M. stenophylla.

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Received 27 February 2006 Revision received 30 May 2006 Accepted 7 June 2006